Showing papers on "Myoglobin published in 2005"
TL;DR: Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers with various grafting ratios were adsorbed to niobium pentoxide-coated silicon wafers and characterized before and after protein adsorption using X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry.
Abstract: Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) copolymers with various grafting ratios were adsorbed to niobium pentoxide-coated silicon wafers and characterized before and after protein adsorption using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Three proteins of different sizes, myoglobin (16 kD), albumin (67 kD), and fibrinogen (340 kD), were studied. XPS was used to quantify the amount of protein adsorbed to the bare and PEGylated surfaces. ToF-SIMS and principal component analysis (PCA) were used to study protein conformational changes on these surfaces. The smallest protein, myoglobin, generally adsorbed in higher numbers than the much larger fibrinogen. Protein adsorption was lowest on the surfaces with the highest PEG chain surface density and increased as the PEG layer density decreased. The highest adsorption was found on lysine-coated and bare niobium surfaces. ToF-SIMS and PCA data evaluation provided further information on the degree of protein denaturation, which, for a particular protein, were found to decrease with increasing PEG surface density and increase with decreasing protein size.
339 citations
TL;DR: High affinity flavoHb and Hb heme ligands, and other inhibitors, may find application as antibiotics and antitumor agents that enhance the toxicity of immune cell-derived *NO or as vasorelaxants that increase *NO signalling.
267 citations
TL;DR: Heme arising from the degradation of hemoglobin, myoglobin, and of enzymes with heme prosthetic groups could be delivered in plasma and the heme trapping mechanisms are summarized.
Abstract: Release of hemoglobin into plasma is a physiological phenomenon associated with intravascular hemolysis. In plasma, stable haptoglobin-hemoglobin complexes are formed and these are subsequently delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis. Heme arising from the degradation of hemoglobin, myoglobin, and of enzymes with heme prosthetic groups could be delivered in plasma. Albumin, haptoglobin, hemopexin, and high and low density lipoproteins cooperate to trap the plasma heme, thereby ensuring its complete clearance. Then hemopexin releases the heme into hepatic parenchymal cells only after internalization of the hemopexin-heme complex by CD91 receptor-mediated endocytosis. Moreover, alpha1-microglobulin contributes to heme degradation by a still unknown mechanism, with the concomitant formation of heterogeneous yellow-brown kynurenine-derived chromophores which are very tightly bound to amino acid residues close to the rim of the lipocalin pocket. During hemoglobin synthesis, the erythroid alpha-chain hemoglobin-stabilizing protein specifically binds free alpha-hemoglobin subunits limiting the free protein toxicity. Although highly toxic because capable of catalyzing free radical formation, heme is also a major and readily available source of iron for pathogenic organisms. Gram-negative bacteria pick up the heme-bound iron through the secretion of a hemophore that takes up either free heme or heme bound to heme-proteins and transports it to a specific receptor, which, in turn, releases the heme and hence iron into the bacterium. Here, hemoglobin and heme trapping mechanisms are summarized.
246 citations
TL;DR: In this article, changes in pigments and color of sardine and mackerel muscles during iced storage were investigated, and myoglobin extraction efficiency depended on fish species, muscle type, storage time and washing process.
215 citations
TL;DR: Heme initiated lipid peroxidation, involved in pathogenesis in humans, is autocatalytic and forms lipid hydroperoxides and is further linked to cross-linking of proteins, which is also important for quality deterioration of muscle-based food.
191 citations
TL;DR: By using time-resolved x-ray crystallography at room temperature, structural relaxations and ligand migration were examined in myoglobin (Mb) mutant L29W from nanoseconds to seconds after photodissociation of carbon monoxide (CO) from the heme iron by Nanosecond laser pulses, thus allowing a quantitative description of the processes involved.
Abstract: By using time-resolved x-ray crystallography at room temperature, structural relaxations and ligand migration were examined in myoglobin (Mb) mutant L29W from nanoseconds to seconds after photodissociation of carbon monoxide (CO) from the heme iron by nanosecond laser pulses. The data were analyzed in terms of transient kinetics by fitting trial functions to integrated difference electron density values obtained from select structural moieties, thus allowing a quantitative description of the processes involved. The observed relaxations are linked to other investigations on protein dynamics. At the earliest times, the heme has already completely relaxed into its domed deoxy structure, and there is no photodissociated CO visible at the primary docking site. Initial relaxations of larger globin moieties are completed within several hundred nanoseconds. They influence the concomitant migration of photodissociated CO to the Xe1 site, where it appears at ≈300 ns and leaves again at ≈1.5 ms. The extremely long residence time in Xe1 as compared with wild-type MbCO implies that, in the latter protein, the CO exits the protein from Xe1 predominantly via the distal pocket. A well-defined deligated state is populated between ≈2 μs and ≈1 ms; its structure is very similar to the equilibrium deoxy structure. Between 1.5 and 20 ms, no CO is visible in the protein interior; it is either distributed among many sites within the protein or has escaped to the solvent. Finally, recombination at the heme iron occurs after >20 ms.
158 citations
TL;DR: Evidence that peroxidase-like enzymic activity, in which a catalytic cycle, driven by peroxides, leads to oxidation of bio molecules, occurs in vivo is reviewed and the role of iron chelators such as desferrioxamine is discussed in terms of their often neglected properties as reducing agents.
Abstract: The heme based respiratory proteins myoglobin and hemoglobin can, under certain conditions, exhibit a peroxidase-like enzymic activity, in which a catalytic cycle, driven by peroxides, leads to oxidation of bio molecules. These heme proteins are implicated in what is termed "oxidative stress" as this catalytic cycle, when it occurs in vivo, generates cytotoxic product that are implicated in the pathology of a number of disease states. Here we review the evidence that such reactions occur in vivo, in particular in animal models and human patients and examine the underlying chemical mechanism. This mechanism involves the production of ferryl heme (Fe(IV)=O(2-)) and it is this and associated radicals that initiate processes such as lipid peroxidation and the generation of bioactive molecules such as isoprostanes. The reactivity of the high oxidation state of the heme also allows us to identify unambiguous biomarkers for its presence in vivo in such conditions as rhabdomyolysis and brain hemorrhage. Ways to inhibit the peroxidatic cycle are discussed and the role of iron chelators such as desferrioxamine is discussed in terms of their often neglected properties as reducing agents. Suppression of the peroxidatic activity of hemoglobin is discussed in the context of the development of blood substitutes.
143 citations
TL;DR: Analysis of the dynamics of the preexisting cavities indicates large fluctuations and frequent connections with the solvent, in agreement with the earlier hypothesis that some of the ligand may escape from the protein through these pathways.
100 citations
TL;DR: To evaluate the sensitivity of the myoglobin and the CK as prognostic tools for the development of Acute renal failure (ARF) and the effect of continuous venovenous haemodiafiltration (CVVHDF) on theMyoglobin elimination in ARF.
Abstract: Background (I) To investigate the kinetics of the myoglobin and creatine kinase (CK) in rhabdomyolysis. Especially to describe those patients in whom an isolated increase in the myoglobin or the CK occurred at a later stage. (II) To evaluate the sensitivity of the myoglobin and the CK as prognostic tools for the development of Acute renal failure (ARF). (III) To investigate the effect of continuous venovenous haemodiafiltration (CVVHDF) on the myoglobin elimination in ARF. Patients and methods Prospective and retrospective cohort study carried out in an ICU of a university hospital. A total of 47 critically ill patients with rhabdomyolysis and a plasma myoglobin > 5000 microg l(-1) were admitted between July 1998 and July 2003. Results (I) The myoglobin peaked 0.66 +/- 0.6 days before the CK. The elimination kinetics of the myoglobin was faster than for the CK. (II) Fifty percent developed ARF. Mortality in the ARF patients was 52% compared to 14% in the non-ARF patients. The sensitivity and specificity of developing ARF were higher with the myoglobin in comparison to the CK. (III) In non-ARF, t(1/2) CK was 25.5 h and t(1/2) myoglobin was 17 h (13-23). In those with ARF treated with CVVHDF, t(1/2) CK was 24.8 and t(1/2) myoglobin was 21 h (17-29). Conclusion (I) The myoglobin peaked earlier than the CK. (II) The myoglobin was a better prognostic tool than the CK. However, the myoglobin also has a wide interindividual range. (III) Though the myoglobin is eliminated in ultrafiltration t(1/2) myoglobin, it was not faster in patients with ARF treated with CVVHDF compared to non-ARF patients.
96 citations
TL;DR: Comparison of the temperature-dependent NO rebinding kinetics of native Mb with that of the bare heme (PPIX) in glycerol reveals that the fast (enthalpically barrierless) No rebinding process observed below 200 K is independent of the presence or absence of the proximal histidine ligand.
Abstract: The rebinding kinetics of NO to the heme iron of myoglobin (Mb) is investigated as a function of temperature. Below 200 K, the transition-state enthalpy barrier associated with the fastest (∼10 ps) recombination phase is found to be zero and a slower geminate phase (∼200 ps) reveals a small enthalpic barrier (∼3 ± 1 kJ/mol). Both of the kinetic rates slow slightly in the myoglobin (Mb) samples above 200 K, suggesting that a small amount of protein relaxation takes place above the solvent glass transition. When the temperature dependence of the NO recombination in Mb is studied under conditions where the distal pocket is mutated (e.g., V68W), the rebinding kinetics lack the slow phase. This is consistent with a mechanism where the slower (∼200 ps) kinetic phase involves transitions of the NO ligand into the distal heme pocket from a more distant site (e.g., in or near the Xe4 cavity). Comparison of the temperature-dependent NO rebinding kinetics of native Mb with that of the bare heme (PPIX) in glycerol re...
95 citations
TL;DR: It is found that the Cu(B) center in cytochrome oxidase has a similar effect on the binding of the diatomics as the distal histidine in myoglobin.
TL;DR: The model proteins cytochrome c, myoglobin, ovalbumin, and β‐lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis and none of the proteins showed any significant Adsorption more than one pH unit above the pIs.
Abstract: The model proteins cytochrome c, myoglobin, ovalbumin, and beta-lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis. The model compounds were selected to cover a wide range of properties. Cytochrome c is a basic protein (isoelectric point (pI): 9.6; M(r): 11.7 kDa), beta-lactoglobulin is rather acidic (pI: 5.4, M(r): 18.4 kDa), myoglobin was chosen as a neutral reference protein (pI: 6.8-7.4, M(r): 17.8 kDa), and ovalbumin (pI: 5.1, M(r): 45.0 kDa) was selected as a relatively larger analyte. First, the pH dependence of adsorption was investigated for the bare fused silica. A clear correlation to the respective pIs was noted. For myoglobin and ovalbumin, none or negligible adsorption was found above the pI, whereas strong adsorption was noted just below this parameter. Cytochrome c and beta-lactoglobulin already showed distinct adsorption above their pIs. However, none of the proteins showed any significant adsorption more than one pH unit above the pIs. For linear polyacrylamide-coated capillaries, a decreased but not a complete lack of adsorption was observed. Here, pH-dependent adsorption was noted as well. Regeneration of the capillaries by rinsing with buffers containing 200 mM SDS was also investigated. This method was completely successful for myoglobin, but that too for only freshly-adsorbed protein. After a storage time of 24 h and due to the aging of the adsorbate, a sufficient regeneration was no longer possible.
TL;DR: The initial results show that it is possible to detect Myoglobin concentrations below 50 ng/mL, which is lower than clinical cut-off for Myoglobin in healthy patients, and the use of SIF-modified substrates is suggested for increasing the sensitivity of surface assays with fluorescence detection.
TL;DR: In this paper, it was shown that even at neutral pH, the heme in hemoglobin is covalently modified by oxidation, and the product, which they termed OxHm, is a green heme with a distinct optical spectrum.
TL;DR: It is reported that lack of myoglobin causes a biochemical shift in cardiac substrate utilization from fatty acid to glucose oxidation and therefore represents a molecular adaptation process allowing to compensate for lack of the cytosolic oxygen carrier myoglobin.
Abstract: Myoglobin is an important intracellular O 2 binding hemoprotein in heart and skeletal muscle. Surprisingly, disruption of myoglobin in mice (myo −/− ) resulted in no obvious phenotype and normal cardiac function was suggested to be mediated by structural alterations that tend to steepen the oxygen pressure gradient from capillary to mitochondria. Here we report that lack of myoglobin causes a biochemical shift in cardiac substrate utilization from fatty acid to glucose oxidation. Proteome and gene expression analysis uncovered key enzymes of mitochondrial β-oxidation as well as the nuclear receptor PPARα to be downregulated in myoglobin-deficient hearts. Using FDG-PET we showed a substantially increased in vivo cardiac uptake of glucose in myo −/− mice (6.7±2.3 versus 0.8±0.5% of injected dose in wild-type, n=5, P 13 C NMR spetroscopic isotopomer studies of isolated hearts which revealed that [1,6- 13 C 2 ]glucose utilization was increased in myo −/− hearts (38±8% versus 22±5% in wild-type, n=6, P 13 C 16 ]palmitate utilization was decreased in the myoglobin-deficient group (42±6% versus 63±11% in wild-type, n=6, P 2 -sparing effect of glucose utilization, the observed shift in substrate metabolism benefits energy homoeostasis and therefore represents a molecular adaptation process allowing to compensate for lack of the cytosolic oxygen carrier myoglobin. Furthermore, our data suggest that an altered myoglobin level itself may be a critical determinant for substrate selection in the heart. The full text of this article is available online at http://circres.ahajournals.org.
TL;DR: In comparisons of anodic and cathodic hemoglobins, heme loss rate better predicted the onset of lipid oxidation than autoxidation rate, suggesting that heme dissociation has a primary role in the ability of different heme proteins to promote lipid oxidation processes.
Abstract: The molecular mass of trout myoglobin was 16017 Da based on electrospray ionization mass spectrometry. A Root effect (low oxygen affinity at pH 6.3) was determined in trout hemoglobin but not myoglobin. At pH 6.3, myoglobin autoxidized more rapidly (3.5-fold) as compared to anodic hemoglobin. Anodic hemoglobin was a better catalyst of lipid oxidation in washed cod muscle as compared to myoglobin at pH 6.3. This suggested that some process other than met heme protein formation was the rate-limiting step in lipid oxidation processes. Heme loss rates were determined using the apomyoglobin mutant H64Y prepared from sperm whale. Anodic hemoglobin released its heme group much more rapidly than myoglobin. In comparisons of anodic and cathodic hemoglobins, heme loss rate better predicted the onset of lipid oxidation than autoxidation rate. These studies collectively suggest that heme dissociation has a primary role in the ability of different heme proteins to promote lipid oxidation processes.
TL;DR: The observed changes indicate that the conversion into fatigue-resistant red fibres depends on the optimum utilization of free fatty acids via albumin transportation, a rearrangement of the creatine kinase isozyme pattern for enhanced mitochondrial activity, and the up-regulation of chaperone-like proteins for stabilising myofibrillar components during the fast-to-slow transition process.
TL;DR: Electrochemical nitric oxide (NO) reduction by heme groups incorporated in films of didodecyldimethylammonium bromide on pyrolitic graphite was investigated and it was shown that DDAB most likely induces the release of the heme group from myoglobin and therefore myoglobin-DDAB and heme-D DAB films give the same voltammetric responses.
Abstract: Electrochemical nitric oxide (NO) reduction by heme groups incorporated in films of didodecyldimethylammonium bromide (DDAB) on pyrolitic graphite was investigated. It is shown that DDAB most likely induces the release of the heme group from myoglobin and therefore myoglobin−DDAB and heme−DDAB films give the same voltammetric responses. This is confirmed by UV/vis spectroscopy showing a clear shift in the Soret band of myoglobin in a DDAB solution. The electrochemical NO reduction on a heme−DDAB film at different pH values reveals the presence of pH-dependent and pH-independent NO reduction pathways. The selectivity of these pathways is probed by combining the rotating ring−disk electrode technique with online electrochemical mass spectroscopy showing that the product of the pH-independent pathway is N2O and the product of the pH-dependent pathway is NH2OH. The preference for one or the other pathway seems to depend on whether a proton or a NO molecule is transferred to a FeII−NO- reaction intermediate an...
TL;DR: Control of O2 versus CO binding in myoglobin (Mb) is tuned by a distal histidine residue through steric and H-bonding interactions through Car-Parrinello DFT calculations, whose efficiency allows full quantum mechanical treatment of the 13 closest residues surrounding the heme.
Abstract: Control of O 2 versus CO binding in myoglobin (Mb) is tuned by a distal histidine residue through steric and H-bonding interactions. These interactions have been evaluated via Car-Parrinello DFT calculations, whose efficiency allows full quantum mechanical treatment of the 13 closest residues surrounding the heme. The small (8°) deviation of the Fe-C-O bond angle from linearity results from the steric influence of a distal valine residue and not the distal histidine. H-bond energies were evaluated by replacing the distal histidine with the non-H-bonding residue isoleucine. Binding energies for CO and O 2 decreased by 0.8 and 4.1 kcal/ mol for MbCO and MbO 2 , in good agreement with experimental H-bond estimates. Ligand discrimination is dominated by distal histidine H-bonding, which is also found to stabilize a metastable side-on isomer of MbO 2 that may play a key role in MbO 2 photodynamics.
TL;DR: This finding confirms that the dephasing of the CO vibration in the native protein is sensitive to the interaction between the ligand and the distal histidine.
Abstract: Ultrafast protein dynamics of the CO adduct of a myoglobin mutant with the polar distal histidine replaced by a nonpolar valine (H64V) have been investigated by spectrally resolved infrared stimulated vibrational echo experiments and molecular dynamics (MD) simulations. In aqueous solution at room temperature, the vibrational dephasing rate of CO in the mutant is reduced by ∼50% relative to the native protein. This finding confirms that the dephasing of the CO vibration in the native protein is sensitive to the interaction between the ligand and the distal histidine. The stimulated vibrational echo observable is calculated from MD simulations of H64V within a model in which vibrational dephasing is driven by electrostatic forces. In agreement with experiment, calculated vibrational echoes show slower dephasing for the mutant than for the native protein. However, vibrational echoes calculated for H64V do not show the quantitative agreement with measurements demonstrated previously for the native protein.
TL;DR: It is demonstrated that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosinelradical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct.
TL;DR: The findings support the use of α-actin as a marker alongside other currently used biological proteins and may be an ideal candidate for the identification of all types of myofibre injury, including sports injuries.
Abstract: Objective: The skeletal muscle protein α-actin was investigated in the serum of subjects with severe skeletal muscle damage to assess its utility as a reliable and predictive marker of muscle damage. Methods: Serum samples were obtained from 33 healthy controls and 33 patients with severe skeletal muscle damage, defined by a total creatine kinase value of >500 IU/l (Rosalki method). Troponin I, troponin T, and myoglobin concentrations were determined by immunoassay and α-actin concentrations by Western blot and densitometry. Results: The mean serum concentration of α-actin in controls and patients with skeletal muscle damage was 600.9 and 1968.51 ng/ml, respectively, a statistically significant difference. Sera of patients with muscle damage showed higher levels of α-actin than of troponin or myoglobin. No significant difference in troponin I levels was observed between the groups. Conclusions: According to these results, α-actin was the most significant skeletal muscle damage marker analysed and may be an ideal candidate for the identification of all types of myofibre injury, including sports injuries. Our findings support the use of α-actin as a marker alongside other currently used biological proteins.
TL;DR: The crystal structure of recombinant D. melanogaster hemoglobin is presented, showing that the protein displays a hexacoordinated heme, whose axial ligands are the proximal and distal His residues, and three protein matrix cavities are spread through the protein matrix.
TL;DR: After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand Rebinding from the solvent.
TL;DR: The concentrations at which desferrioxamine inhibits the formation of heme to protein cross-linked myoglobin and prevents the pro-oxidant activity of native and oxidatively modified myoglobins are comparable to the concentrations used for in vivo studies of iron-related oxidative stress, indicating that the ameliorative effects of treatment of posthemolytic events by desferRIoxamine cannot be exclusively assigned to its ability to chelate free iron.
Abstract: The heme group of myoglobin can form a covalent bond to the protein when met (ferric) myoglobin is reacted with peroxides under acidic conditions. This heme to protein cross-linked species is highly pro-oxidant and found in the urine of patients with rhabdomyolytic-associated acute renal failure. Desferrioxamine, an iron-chelating agent used in the treatment of iron overload, is reported to be partially effective at preventing kidney failure following rhabdomyolysis. In this article, we show that in addition to its capacity as an iron chelator, desferroxamine can inhibit the peroxide-induced formation of heme to protein cross-linked myoglobin and decreases the pro-oxidant activity of both native and heme to protein cross-linked myoglobin. The mechanism of peroxidation and of heme to protein cross-linking involves the formation of ferryl intermediate (Fe(4+)=O(2-)), and it is by the reduction of this intermediate to the ferric form that desferrioxamine can exert inhibitory effects. The concentrations at which desferrioxamine inhibits the formation of heme to protein cross-linked myoglobin and prevents the pro-oxidant activity of native and oxidatively modified myoglobins are comparable to the concentrations used for in vivo studies of iron-related oxidative stress. Thus, the ameliorative effects of treatment of posthemolytic events by desferrioxamine cannot be exclusively assigned to its ability to chelate free iron.
TL;DR: Eight X-ray structures of myoglobin mutant L29W at various experimental conditions are determined, and two distinct conformations of the CO-ligated protein were identified, giving rise to two stretching bands of heme-bound CO.
Abstract: We have determined eight X-ray structures of myoglobin mutant L29W at various experimental conditions. In addition, infrared spectroscopic experiments are presented, which are discussed in the light of the X-ray structures. Two distinct conformations of the CO-ligated protein were identified, giving rise to two stretching bands of heme-bound CO. If L29W MbCO crystals are illuminated around 180 K, a deoxy species is formed. The CO molecules migrate to the proximal side of the heme and remain trapped in the so-called Xe1 cavity upon temperature decrease to 105 K. The structure of this photoproduct is almost identical to the equilibrium high-temperature deoxy Mb structure. If the temperature is cycled to increasingly higher values, CO recombination is observed. Three intermediate structures have been determined during the rebinding process. Efficient recombination occurs only above 180 K, the characteristic temperature for the onset of protein dynamics. Rebinding is remarkably slow because bulky residues His...
01 Jan 2005
TL;DR: Dimethyl-1-pyrroline N-oxide spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation as discussed by the authors.
Abstract: Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO. Published by Elsevier Inc.
TL;DR: Horse myoglobin was less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments, suggesting helix propensity is an important component of the explanation for the difference in stability between the two proteins.